LOCUS       Exported                5897 bp ds-DNA     circular SYN 15-JUN-2021
DEFINITION  Expresses both yeast Tim9 and yeast Tim10 (the latter with cleavable
            N-ter His-tag), codon-optimized for E. coli production.
ACCESSION   .
VERSION     .
KEYWORDS    pETDuet-1 TIM9,10
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5897)
  AUTHORS   Weinhaupl K, Lindau C, Hessel A, Wang Y, Schutze C, Jores T, 
            Melchionda L, Schonfisch B, Kalbacher H, Bersch B, Rapaport D, 
            Brennich M, Lindorff-Larsen K, Wiedemann N, Schanda P
  TITLE     Structural Basis of Membrane Protein Chaperoning through the 
            Mitochondrial Intermembrane Space.
  JOURNAL   Cell. 2018 Nov 15;175(5):1365-1379.e25. doi: 
            10.1016/j.cell.2018.10.039.
  PUBMED    30445040
REFERENCE   2  (bases 1 to 5897)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jun 15, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5897
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             130..150
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     promoter        501..519
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    520..544
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             863..907
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(945..963)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      959..1006
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1043..1498
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1130..1149)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1340..1361
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1613..2473)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2236..2255
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2474..2566)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2647..3235
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3136..3155
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3389..3406
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3421..3560
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3486..3505)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3646..3668
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3662..3853)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4425..5507)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5469..5488
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5508..5585)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5791..5810
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcaccatca tcaccacagc
      121 caggatccgg agaatttgta ttttcagggt agctttttgg gtttcggcgg cggtcagcca
      181 caattgtctt cccagcaaaa gatccaagca gcggaagcgg aattggatct ggtgacggat
      241 atgtttaaca aactggtgaa taactgctac aagaaatgta tcaacacttc ctatagcgag
      301 ggtgagttga ataagaacga gagcagctgt ctggaccgtt gtgttgccaa gtattttgag
      361 accaacgtcc aggtcggcga gaacatgcaa aaaatgggtc aatcgttcaa tgccgcgggt
      421 aagttttaaa agcttgcggc cgcataatgc ttaagtcgaa cagaaagtaa tcgtattgta
      481 cacggccgca taatcgaaat taatacgact cactataggg gaattgtgag cggataacaa
      541 ttccccatct tagtatatta gttaagtata agaaggagat atacatatgg atgcattgaa
      601 ctctaaagaa caacaggaat ttcagaaagt tgtggaacaa aaacagatga aggatttcat
      661 gcgtctgtat tccaacttgg tagagcgttg tttcaccgac tgtgtgaatg acttcaccac
      721 atcaaagctg accaacaaag aacagacttg tatcatgaaa tgcagcgaga agtttctgaa
      781 acactccgag cgcgtgggtc aacgttttca ggaacaaaat gcggcgctgg gccagggtct
      841 gggtcgttaa ctcgagtctg gtaaagaaac cgctgctgcg aaatttgaac gccagcacat
      901 ggactcgtct actagcgcag cttaattaac ctaggctgct gccaccgctg agcaataact
      961 agcataaccc cttggggcct ctaaacgggt cttgaggggt tttttgctga aaggaggaac
     1021 tatatccgga ttggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     1081 gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct
     1141 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     1201 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag
     1261 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     1321 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc
     1381 tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat
     1441 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttct
     1501 ggcggcacga tggcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     1561 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     1621 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     1681 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     1741 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     1801 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     1861 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     1921 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     1981 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     2041 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     2101 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     2161 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     2221 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     2281 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     2341 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     2401 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     2461 gttgaatact catactcttc ctttttcaat catgattgaa gcatttatca gggttattgt
     2521 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg tcatgaccaa
     2581 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     2641 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     2701 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     2761 tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca
     2821 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     2881 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     2941 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     3001 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     3061 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     3121 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     3181 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     3241 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     3301 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
     3361 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaagagcg
     3421 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca atggtgcact
     3481 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg ctatcgctac
     3541 gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     3601 cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt
     3661 gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa agctcatcag
     3721 cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc tcgttgagtt
     3781 tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg gcggtttttt
     3841 cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg gtaatgatac
     3901 cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat gcccggttac
     3961 tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag agaaaaatca
     4021 ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag ggtagccagc
     4081 agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc cgcgtttcca
     4141 gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc gcagacgttt
     4201 tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc taaccagtaa
     4261 ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg ctagtcatgc
     4321 cccgcgccca ccggaaggag ctgactgggt tgaaggctct caagggcatc ggtcgagatc
     4381 ccggtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg cccgctttcc
     4441 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg
     4501 gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg caacagctga
     4561 ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct ggtttgcccc
     4621 agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga gctgtcttcg
     4681 gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc gcagcccgga ctcggtaatg
     4741 gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt gggaacgatg
     4801 ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca gtcgccttcc
     4861 cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc agccagacgc
     4921 agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg gtgacccaat
     4981 gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat aatactgttg
     5041 atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca ggcagcttcc
     5101 acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact gacgcgttgc
     5161 gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc taccatcgac
     5221 accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac aatttgcgac
     5281 ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg tttgcccgcc
     5341 agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc ttccactttt
     5401 tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac ggtctgataa
     5461 gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt caccaccctg
     5521 aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgcg ccattcgatg
     5581 gtgtccggga tctcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     5641 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     5701 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     5761 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     5821 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tcgagatcga
     5881 tctcgatccc gcgaaat
//
